The principal research goal of the Synaptic Function Unit is to understand the molecular mechanisms underlying the neurotransmitter release process and its modulation. In past year, we have obtained promising results on following three projects: (1) Cloning and characterization of Snapin, a novel SNARE-associated protein implicated in synaptic transmission. Using the yeast two-hybrid system, we identified a novel brain-specific protein family called Snapin which is associated with the synaptic vesicle docking/fusion SNARE complex. We demonstrated that Snapin is enriched in neurons and is exclusively located on synaptic vesicle membranes, indicating its role in vesicle docking or fusion. Coincubation of Snapin peptides corresponding to the SNAP-25-binding site blocks the association of the SNARE complex with Synaptotagmin - a probable candidate for the Ca2+-sensor involved in fast, Ca2+-dependent, neurotransmitter release. Introducing truncated Snapin and peptides containing the SNAP-25 binding sequence into presynaptic superior cervical ganglion neurons (SCGNs) in culture reversibly inhibits synaptic transmission, while heat-denatured Snapin or peptides with point mutations and scrambled sequences have no effect. We also identified that several residues on Snapin's carboxyl terminal that are critical for its binding to SNAP-25. These residues also have physiologic roles in neurotransmission. More recent experiments on Xenopus neuron-muscle cocultures have shown that presynaptic loading of full-length Snapin results in a 2-fold increase in both spontaneous synaptic current (SSC) and evoked synaptic current (ESC), suggesting that synaptic efficacy can be boosted by a Snapin-dependent mechanism and that Snapin may be a limiting component of such a mechanism. Recently, it has been demonstrated that both recombinant and native Snapin isolated from synaptosomes are phosphorylated by PKA and PKC, suggesting Snapin might be another target for modulating transmitter release as well as neuronal plasticity via a second messenger signal transduction pathway. Together, our results suggest Snapin plays a key regulator role at a step between vesicle docking and neurotransmitter release. It does this by perhaps potentiating the interaction of synaptotagmin with the SNAREs, resulting in the final fusion step. (The paper on Snapin has been submitted). (2) Cloning and biochemical characterization of an unknown syntaxin-interacting protein. Using the yeast two-hybrid system we also cloned an uncharacterized syntaxin-binding protein from a human brain cDNA library. Northern and immunoblotting analysis demonstrated that it is exclusively expressed in brain and located in presynaptic cytosolic fraction. Its specific interaction with syntaxin was confirmed in both in vitro binding assays with recombinant syntaxin and immunoprecipitation studies with native syntaxin from rat synaptosomes. It is composed of an amino terminal coiled coil region, a conserved domain presented in different families of vesicular fusion proteins. The in vitro binding studies showed that the coiled coil domain is required for interaction with syntaxin. (3) Biochemical characterization of presynaptic proteins in BDNF knockout mice (Collaboration with Bai Lu's group). BDNF promotes LTP at hippocampal CA1 synapses by a presynaptic enhancement of synaptic transmission during high-frequency stimulation. With biochemical approach we found that hippocampal synaptosomes prepared from BDNF mutant mice exhibited a marked decrease in the levels of synaptophysin as well as synaptobrevin (VAMP-2), a protein known to be involved in vesicle docking and fusion. Quantitative analysis indicated that the level of synaptophsyin and VAMP-2 in +/- hippocampus were reduced by more than 72% and 82 %, respectively. Other synaptic proteins including synaptotagmin, syntaxin and SNAP-25 were unaffected. Application of BDNF on the hippocampal slices from mutant mice restores both proteins to normal levels. Our results support the physiological and morphological findings of Bai Lu's group, and suggest a novel role for BDNF in the mobilization and/or docking of synaptic vesicles to presynaptic active zones. (The paper has been submitted). Currently, we are working to further characterize the roles of Snapin and the new syntaxin-binding protein during the process of synaptic vesicle docking and fusion, using combined approaches including molecular (gene knockout), biochemical (phosphorylation modulation) and electrophysiological studies (in both synapses formed between cultured SCGNs and between cultured Xenopus nerve-muscle).